Nonfimbrial, mannose-resistant hemagglutinins (nonfimbrial adhesions [NFA] NFA-1 and NFA-2) were extracted from two agar-grown urinary isolates of Escherichia coli strains 827 (O83:K1:H4) and 54 (O14:K?:H11). The proteins were purified to homogeneity by ammonium sulfate precipitation and column chromatography. Nonfimbrial adhesins are soluble proteins, which tend to form aggregates of molecular weight above 10(6). NFA-1 and NFA-2 consist of subunits of 21,000 and 19,000 molecular weight, respectively. Both hemagglutinins caused hemagglutination of human erythrocytes and bound to human kidney cell monolayers. The binding of bacteria and hemagglutinins was assessed by using suitable antisera as detectors in an enzyme-linked immunosorbent assay. NFA-1 and NFA-2 inhibited the adherence of their respective strains to human kidney cells in a linear dose response. NFA-2 also inhibited heterologous strain adherence, but NFA-1 did not. Hemabsorption of bacterial suspension with erythrocytes at 4 degrees C, followed by differential centrifugation, enabled us to obtain a bacterial suspension lacking nonfimbrial adhesins in the supernatant and an adhesin-enriched bacterial suspension that was eluted from erythrocytes at 40 degrees C. Bacteria eluted from erythrocytes exhibited a higher adherence capacity than unfractionated cells. Bacteria of the fraction lacking adhesins did not adhere to human kidney cells. Electron microscope examinations showed the presence of an extracellular capsule-like layer in adhering E. coli 827, but not in nonadhering bacteria. E. coli 54 did not express the adhesin as a capsule. We conclude that E. coli 827 and 54 produce extracellular adhesins consisting of soluble proteins which are differently expressed and antigenically distinct. The adhesins seem to share a common receptor and mediate the adherence of two uropathogenic E. coli strains to epithelial cells.